The Aim of this proposal is to study the function of transcriptional repressor of the Hes and Hey family in supporting cell development and their potential involvement in repressing hair cell regeneration. Mammals do not regenerate lost hair cells and hair cell loss is a leading cause for deafness in humans. In contrast, supporting cells in birds can function as hair cell progenitors and are capable to regenerate lost hair cells. The choice whether to differentiate into a hair cell or a supporting cell, is controlled by Notch signaling. In the inner ear Notch signaling is believed to be mediated by Hes1 and Hes5, two transcriptional repressors belonging to hairy and enhancer of split (HES) family. We recently discovered that HES related Hey genes (Hey1, Hey2, HeyL) are co-expressed with Hes1 and Hes5 in differentiating supporting cells. In Specific Aim 1 we will address the question if the Hes and Hey genes function synergistically to maintain a supporting cells specific state. We will generate based on their expression pattern combinations of Hey Hes mutant mice and analyze the extent of supporting cell to hair cell trans-differentiation in the single mutant and double mutant mice. In Specific Aim 2 we propose to determine if the persistence of Hey and Hes gene expression in the damaged organ of Corti blocks hair cell regeneration. We propose to analyze if Hes and Hey mutant supporting cells have an enhanced capacity to generate hair cells in primary cell culture. Further we propose to test if loss of Hes or Hey function is sufficient to induce hair cell regeneration in the damaged cochlear using organ culture. In this grant proposal, we want to investigate the role of transcriptional repressors of the Hes and Hey gene family in inhibiting hair cell regeneration. Sensory hair cell loss in the inner ear is a leading cause of deafness and balance disorders. Understanding the molecular mechanisms preventing hair cell regeneration in mammals is crucial for developing successful hair cell replacement therapies.